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The sensitivity and specificity of three fluorescent probes used for cytofluorimetric analysis of mitochondrial membrane potential (ΔΨ) were studied in the U937 human cell line. First, the role of plasmamembrane in influencing the binding of the probes to mitochondria has been investigated. The depolarization of plasmamembrane with high doses of extracellular KCl had no immediate effects on the loading of JC-1, DiOC₆(3) and rhodamine 123 (R123). However, after a few hours of culture in the presence of KCl, significant changes were observed only in cells stained with DiOC₆(3). Second, a comparative study was performed concerning the effects of agents capable of collapsing ΔΨ. While adding FCCP to cell cultures resulted in consistent changes in the fluorescence emission of both JC-1 and DiOC₆(3) – but not of R123 – only cells stained with JC-1 responded to valinomycin. On the whole, our data indicate that …